CN115326983A - Method for measuring contents of various components in vital qi tablet extract - Google Patents
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Abstract
The invention provides a method for measuring the contents of various components in a vital qi tablet extract, which comprises the following steps: dissolving the extract of the Zhengqi tablet in a solvent, performing ultrasonic extraction and filtration, detecting the obtained test solution by using an ultra-high performance liquid chromatography, and determining 4 index components in the test solution: content of liquiritin, hesperidin, honokiol, and magnolol. The method for measuring the contents of various components in the vital qi tablet extract provided by the invention has the advantages of good linear relation, good repeatability, good precision, high accuracy and good stability, can be used for data accumulation of multiple batches of samples, establishes a reasonable content limit, and ensures stable production and clinical curative effect.
Description
Technical Field
The invention belongs to the technical field of traditional Chinese medicine component detection, relates to a method for determining the content of various components in a vital qi tablet extract, and particularly relates to a method for determining the content of 4 components in the vital qi tablet extract: UPLC detection method for content of liquiritin, hesperidin, honokiol and magnolol.
Background
The Zhengqi tablet is prepared from 10 Chinese medicinal materials including patchouli oil, perilla leaf oil, aucklandia root, atractylodes root, licorice root, poria, tangerine peel, pinellia tuber, ginger-processed magnolia bark and ginger, and is loaded into the first part of the pharmacopoeia of the people's republic of China 2020 edition. The Zhengqi tablet extract is used as a midbody of the Zhengqi tablet and is formed by decocting and concentrating six ingredients of liquorice, tuckahoe, dried orange peel, processed pinellia tuber, ginger officinal magnolia bark, ginger and the like by using water, and the content determination method of the existing Zhengqi tablet extract is lacked, so that a lot of inconvenience is caused to the production quality control.
Disclosure of Invention
In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide a method for measuring the contents of multiple components in a healthy qi tablet extract, which is used to solve the problem of the prior art that the quality of the healthy qi tablet extract is not effectively controlled by measuring the contents of multiple components in the healthy qi tablet extract.
In order to achieve the above and other related objects, a first aspect of the present invention provides a method for determining the contents of a plurality of components in a healthy qi tablet extract, comprising: dissolving the extract of the Zhengqi tablet in a solvent, performing ultrasonic extraction, filtering, detecting the obtained test solution by using ultra-performance liquid chromatography (UPLC), and determining 4 index components in the test solution: content of liquiritin, hesperidin, honokiol, and magnolol.
Preferably, the CAS number of the liquiritin is 551-15-5, the CAS number of the hesperidin is 520-26-3, the CAS number of the honokiol is 35354-74-6, and the CAS number of the magnolol is 528-43-8.
The preparation process of the Zhengqi tablet extract is obviously different from that of the Zhengqi tablet. Specifically, the Zhengqi tablet extract is obtained by decocting liquorice, poria cocos, dried orange peel, processed pinellia ternate, ginger-processed magnolia bark and ginger twice with water, decocting 8 times of water for 3 hours for the first time, decocting 6 times of water for 2 hours for the second time, filtering decoction, and concentrating under reduced pressure until the relative density is 1.03-1.05 (50-70 ℃). The ZHENGQI tablet is prepared from ZHENGQI tablet powder (pulverized mixture of radix aucklandiae, rhizoma Atractylodis, glycyrrhrizae radix, and sucrose), perilla leaf oil and oleum herba Pogostemonis by granulating with fluidized bed drying machine, adding pulvis Talci, and pressing.
The Zhengqi tablet extract is used as a Zhengqi tablet intermediate and is prepared by decocting and concentrating six ingredients of liquorice, tuckahoe, dried orange peel, processed pinellia tuber, ginger officinal magnolia bark, ginger and the like. Modern pharmacological research shows that magnolol and honokiol are main components of traditional Chinese medicine magnolia officinalis and have strong central inhibition effect; hesperidin represents the main chemical component flavonoid in pericarpium citri reticulatae, and has the effects of resisting shock, resisting arteriosclerosis, soothing liver and gallbladder, resisting allergy, resisting bacteria and the like; liquiritin represents a main chemical component flavonoid compound in liquorice, and has the effects of resisting infection, virus and the like. Therefore, the invention selects liquiritin, hesperidin, honokiol and magnolol as index components of the vital qi tablet extract.
Preferably, the solvent is methanol.
Preferably, the ratio of the mass g of the n-qi tablet extract to the volume mL of the solvent is 1.
Preferably, the time of ultrasonic extraction is 20 to 40 minutes, preferably 30 minutes.
Preferably, the power of the ultrasonic extraction is 200-300W, preferably 250W; the frequency of the ultrasonic extraction is 30-50 kHz, preferably 40kHz.
Preferably, the water temperature in the ultrasonic instrument is controlled to be less than or equal to 30 ℃ before the ultrasonic extraction, and the water temperature in the ultrasonic instrument is controlled to be less than or equal to 45 ℃ after the ultrasonic extraction.
Preferably, the ultrasonic extraction is followed by cooling. And cooling to room temperature, wherein the room temperature is 20-30 ℃.
Preferably, the filtration is a supernatant filtration membrane, and after discarding the primary filtrate, a subsequent filtrate is taken.
Preferably, the filter is a 0.22 μm filter.
Preferably, the detection is performed by Ultra Performance Liquid Chromatography (UPLC), comprising the steps of:
1) Preparing a reference solution: adding solvent into glycyrrhizin, hesperidin, honokiol and magnolol as reference substances, dissolving, and diluting to desired volume to obtain reference substance solution;
2) Sample detection: respectively detecting the test solution and the reference solution in the step 1) by adopting Ultra Performance Liquid Chromatography (UPLC), comparing the retention time for qualitative determination, and determining the content of liquiritin, hesperidin, honokiol and magnolol in the test solution by adopting an external standard method for quantitative determination.
Preferably, in step 1), the control solution is shaken up and filtered, and the filtrate is taken out.
Preferably, in step 1), the reference solution is prepared by stepwise dilution.
Preferably, in the step 1), the content range of the liquiritin in the reference solution is 0.90-45.22 μ g/ml, the content range of the hesperidin is 4.75-237.61 μ g/ml, the content range of the honokiol is 0.99-49.60 μ g/ml, and the content range of the magnolol is 0.91-45.40 μ g/ml.
More preferably, the content range of the liquiritin in the control solution is 8 mu g/ml, the content range of the hesperidin is 80 mu g/ml, the content range of the honokiol is 8 mu g/ml, and the content range of the magnolol is 8 mu g/ml.
Preferably, in step 1), the solvent is methanol.
Preferably, in the step 2), the detector is a Diode Array Detector (DAD) in the Ultra Performance Liquid Chromatography (UPLC).
Preferably, in the step 2), the chromatographic column in the ultra-high performance liquid chromatography is a C18 chromatographic column.
More preferably, the chromatographic column in the ultra-high performance liquid chromatography is a WatersAcquity UPLC BEH C18 chromatographic column (column length is 100mm, inner diameter is 2.1mm, and packing particle size is 1.7 μm).
Preferably, in the step 2), the detection wavelength in the ultra-high performance liquid chromatography is 220 to 230nm. More preferably, the detection wavelength is 225nm.
Preferably, in the step 2), the column temperature in the ultra-high performance liquid chromatography is 30-50 ℃. More preferably, the column temperature in the ultra-high performance liquid chromatography is 40 ℃.
Preferably, in the step 2), the flow rate in the ultra high performance liquid chromatography is 0.1 to 0.3mL/min. More preferably, the flow rate in the ultra high performance liquid chromatography is 0.2mL/min.
Preferably, in the step 2), the sample amount in the ultra high performance liquid chromatography is 0.5 to 5 μ L. More preferably, the sample amount in the ultra high performance liquid chromatography is 1 μ L.
Preferably, in the step 2), in the ultra-high performance liquid chromatography, the mobile phase is acetonitrile-0.05-0.15% phosphoric acid aqueous solution, wherein the phase A is acetonitrile, and the phase B is 0.05-0.15% phosphoric acid aqueous solution; the analysis time is 34min; and (4) gradient elution. More preferably, in the ultra-high performance liquid chromatography, the mobile phase is acetonitrile-0.10% phosphoric acid aqueous solution, wherein the phase A is acetonitrile, and the phase B is 0.10% phosphoric acid aqueous solution; the analysis time is 34min; gradient elution.
The 0.05 to 0.15 percent phosphoric acid aqueous solution is 0.05 to 0.15 percent phosphoric acid aqueous solution by volume percentage. The 0.10% phosphoric acid aqueous solution is 0.10% by volume phosphoric acid aqueous solution.
More preferably, the specific procedure for the gradient elution is shown in table 1 as:
0-5min, phase A: the volume ratio of the phase B is 17:83-17:83;
5-7min, phase A: the volume ratio of the phase B is 17:83-20:80;
7-11min, phase A: the volume ratio of the phase B is 20:80-40:60;
11-12min, phase A: the volume ratio of the phase B is 40:60-55:45, a first step of;
12-22min, phase A: the volume ratio of the phase B is 55:45-58:42;
22-23min, phase A: the volume ratio of the phase B is 58:42-95:5;
23-28min, phase A: the volume ratio of the phase B is 95:5-95:5;
28-29min, phase A: the volume ratio of the phase B is 95:5-17:83;
29-34min, phase A: the volume ratio of the phase B is 17:83-17:83.
the analysis time of the determination method is very short at 34min, various components in the vital qi tablet extract can be rapidly determined, and 4 components are detected in the first 20 min, so that the reagent is saved, and the daily detection work of QC can be greatly reduced.
TABLE 1
Preferably, in step 2), the external standard method comprises the following steps:
a) Preparing a series of reference substance solutions with different concentrations according to the step 1), respectively carrying out UPLC detection to obtain linear relations between chromatographic peak areas of the liquiritin, the hesperidin, the honokiol and the magnolol and contents of the liquiritin, the hesperidin, the honokiol and the magnolol, drawing corresponding standard working curves, and respectively calculating regression equations of the standard working curves of the liquiritin, the hesperidin, the honokiol and the magnolol;
b) And (3) carrying out UPLC detection on the test solution, substituting the chromatographic peak areas of the obtained liquiritin, hesperidin, honokiol and magnolol into the regression equation of the standard working curve of the corresponding liquiritin, hesperidin, honokiol and magnolol in the step A), and calculating to obtain the content of the liquiritin, hesperidin, honokiol and magnolol in the test solution.
More preferably, in the standard working curve, the chromatographic peak areas of liquiritin, hesperidin, honokiol and magnolol are used as ordinate (Y axis), and the contents (i.e. concentrations) of the corresponding liquiritin, hesperidin, honokiol and magnolol are used as abscissa (X axis).
The second aspect of the invention provides application of a method for measuring the content of multiple components in a vital qi tablet extract in quality detection of the vital qi tablet extract.
As mentioned above, the method for determining the contents of various components in the zhengqi tablet extract provided by the invention adopts the pretreatment and instrument detection methods of optimized conditions, and the method comprises the following steps of: and carrying out accurate quantitative and qualitative detection on liquiritin, hesperidin, honokiol and magnolol. The method can be used for analyzing 4 active ingredients in the qiqi tablet extract in a short time: separating and measuring liquiritin, hesperidin, honokiol and magnolol. The method is simple to operate and convenient to control, the standard curves of the 4 measured components have good linear relation in respective ranges, good repeatability, good precision, high accuracy and good stability, and can be used for data accumulation of multiple batches of samples, a reasonable content limit is formulated, and stable production and clinical curative effect are ensured.
Drawings
FIG. 1 shows the chromatogram for investigating the specificity of liquiritin in the present invention.
Fig. 2 shows a specificity investigation chromatogram of hesperidin in the present invention.
Figure 3 shows the specificity survey chromatogram of honokiol and magnolol in the present invention.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are only intended to illustrate the invention and are not intended to limit the scope of the invention.
The following embodiments of the present invention are provided by way of specific examples, and other advantages and effects of the present invention will be readily apparent to those skilled in the art from the disclosure herein. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
The reagents and equipment used in the following examples are as follows:
1. reagent
25 batches of positive air tablet extract (batch Nos. S1-210102, S2-210103, S3-210104, S4-210106, S5-210107, S6-210108, S7-210109, S8-210201, S9-210202, S10-210702, S11-210703, S12-210901, S13-210902, S14-210903, S15-220601, S16-220602, S17-220603, S151-220708, prepared by Shanghai and yellow drug industries, ltd.); negative samples (lacking licorice healthy qi tablet extract, lacking dried orange peel healthy qi tablet extract and lacking magnolia officinalis healthy qi tablet extract) are provided by Shanghai and Huangyao limited company.
Comparison products: liquiritin (batch No. 111610-201908, mass fraction of 95%), hesperidin (batch No. 110721-201818, mass fraction of 96.2%), honokiol (batch No. 110730-201915, mass fraction of 99.8%), magnolol (batch No. 110729-201513, mass fraction of 98.8% -99%), all of which were purchased from the institute of food and drug testing in china.
Reagent: methanol (analytical pure AR, national chemical group, ltd); acetonitrile (chromatographically pure, TEDIA corporation, usa); phosphoric acid (chromatographically pure, TEDIA corporation, usa); ultrapure water was prepared by a Milli-Q ultrapure water treatment system.
2. Instrument
A Waters Acquity UPLC H-Class ultra-high performance liquid chromatograph (equipped with an Empower 3 chromatographic workstation, a quaternary ultra-high pressure solvent manager, an automatic sample introduction sample manager, a column incubator, a PDAe lambda detector, and Waters corporation, USA); SB-5200DTD ultrasonic cleaning machine (Ningbo Xinzhi Biotech Co., ltd.); analytical electronic balances of models AL204 and XS205 (Metler-Tolydo Meter Shanghai Co., ltd.); milli-QAdvantage A10 ultra pure water system (Merck Millipore, germany).
Example 1
1. Preparation of test solution
1.0g of the extract sample of the Zhengqi tablet is precisely weighed, placed in a 50mL centrifuge tube, precisely added with 20mL of methanol, ultrasonically extracted (250w, 40kHz) for 30 minutes, the water temperature before ultrasonic cannot exceed 30 ℃, and the water temperature after ultrasonic is not higher than 45 ℃ basically. Cooling, filtering the supernatant with 0.22 μm filter membrane, and collecting the filtrate to obtain sample solution No. 1.
2. Preparation of control solutions
Precisely weighing glycyrrhizin, hesperidin, honokiol and magnolol as reference substances, adding methanol, dissolving in a 100mL volumetric flask to constant volume, and preparing into reference substance stock solution. The reference stock solution was stored in a refrigerator at 4 ℃ in the dark.
And diluting the reference substance stock solution step by adopting methanol and fixing the volume to a certain volume to prepare a series of reference substance solutions with different concentrations. In a series of reference substance solutions with different concentrations, the content range of liquiritin is 0.90-45.22 mu g/ml, the content range of hesperidin is 4.75-237.61 mu g/ml, the content range of honokiol is 0.99-49.60 mu g/ml, and the content range of magnolol is 0.91-45.40 mu g/ml. Shaking the control solution, filtering, and collecting the filtrate.
3. Measurement of
Detecting the sample solution 1# and a series of reference solutions with different concentrations respectively by Ultra Performance Liquid Chromatography (UPLC), comparing retention time for qualitative determination, and quantifying by external standard method. A series of reference substance solutions with different concentrations are respectively subjected to UPLC detection to obtain linear relations between chromatographic peak areas of the liquiritin, the hesperidin, the honokiol and the magnolol and the concentrations of the corresponding liquiritin, the hesperidin, the honokiol and the magnolol, corresponding standard working curves are drawn, and regression equations of the standard working curves of the liquiritin, the hesperidin, the honokiol and the magnolol are respectively obtained through calculation. And performing UPLC detection on the test solution 1#, substituting the chromatographic peak areas of the obtained liquiritin, hesperidin, honokiol and magnolol into the regression equation of the standard working curve of the corresponding liquiritin, hesperidin, honokiol and magnolol, and calculating to obtain the concentrations of the liquiritin, hesperidin, honokiol and magnolol in the test solution 1#.
The high performance liquid chromatography comprises the following detection conditions:
the detector is a Diode Array Detector (DAD); the chromatographic column is a WatersAcquity UPLC BEH C18 chromatographic column (the column length is 100mm, the inner diameter is 2.1mm, and the particle size of the filler is 1.7 mu m); the detection wavelength is 225nm; the column temperature was 40 ℃; the flow rate is 0.2mL/min; the sample amount is 1 mul; the mobile phase is acetonitrile-0.10% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.10% phosphoric acid water solution; the analysis time is 34min; gradient elution.
The specific procedure for gradient elution was:
0-5min, phase A: the volume ratio of the phase B is 17:83-17:83;
5-7min, phase A: the volume ratio of the phase B is 17:83-20:80;
7-11min, phase A: the volume ratio of the phase B is 20:80-40:60;
11-12min, phase A: the volume ratio of the phase B is 40:60-55:45, a first step of;
12-22min, phase A: the volume ratio of the phase B is 55:45-58:42;
22-23min, phase A: the volume ratio of the phase B is 58:42-95:5;
23-28min, phase A: the volume ratio of the phase B is 95:5-95:5;
28-29min, phase A: the volume ratio of the phase B is 95:5-17:83;
29-34min, phase A: the volume ratio of the phase B is 17:83-17:83.
example 2
1. Preparation of test solution
Taking 1.0g of a Zhengqi tablet extract sample, precisely weighing, placing in a 50mL centrifuge tube, precisely adding 15mL of methanol, performing ultrasonic extraction (240w, 35kHz) for 40 minutes, wherein the water temperature before ultrasonic extraction cannot exceed 30 ℃, and the water temperature after ultrasonic extraction is not higher than 45 ℃ basically. Cooling, filtering the supernatant with 0.22 μm filter membrane, and collecting the filtrate to obtain sample solution 2#.
2. Preparation of control solutions
Precisely weighing glycyrrhizin, hesperidin, honokiol and magnolol as reference substances, adding methanol, dissolving in a 100mL volumetric flask to constant volume, and preparing into reference substance stock solution. The reference stock solution was stored in a refrigerator at 4 ℃ in the dark. And diluting the reference substance stock solution step by adopting methanol and fixing the volume to a certain volume to prepare a series of reference substance solutions with different concentrations.
In a series of control solutions with different concentrations, the concentration ranges of liquiritin, hesperidin, honokiol and magnolol are the same as the step 2 in the example 1.
3. Measurement of
Respectively detecting the sample solution 2# and a series of reference substance solutions with different concentrations by Ultra Performance Liquid Chromatography (UPLC), comparing retention time for qualitative determination, and quantifying by external standard method to obtain the concentrations of liquiritin, hesperidin, honokiol and magnolol in the sample solution 2#. The specific quantitative procedure was the same as in step 3 of example 1.
Wherein, the high performance liquid chromatography comprises the following detection conditions:
the detector is a Diode Array Detector (DAD); the chromatographic column is a WatersAcquity UPLC BEH C18 chromatographic column (the column length is 100mm, the inner diameter is 2.1mm, and the particle size of the filler is 1.7 mu m); the detection wavelength is 220nm; the column temperature is 35 ℃; the flow rate is 0.1mL/min; the sample injection amount is 0.5 mu L; the mobile phase is acetonitrile-0.15% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.15% phosphoric acid water solution; the analysis time is 34min; and (4) gradient elution.
The procedure for gradient elution was the same as in step 3 of example 1.
Example 3
1. Preparation of test solution
1.0g of Zhengqi tablet extract sample is precisely weighed, placed in a 50mL centrifuge tube, precisely added with 25mL of methanol, ultrasonically extracted (260w, 45kHz) for 20 minutes, wherein the water temperature before ultrasonic treatment cannot exceed 30 ℃, and the water temperature after ultrasonic treatment is basically not higher than 45 ℃. Cooling, filtering the supernatant with 0.22 μm filter membrane, and collecting the filtrate to obtain test solution # 3.
2. Preparation of control solutions
Precisely weighing glycyrrhizin, hesperidin, honokiol and magnolol as reference substances, adding methanol, dissolving in a 100mL volumetric flask to constant volume, and preparing into reference substance stock solution. The control stock solution was stored in a refrigerator at 4 ℃ protected from light. And diluting the reference substance stock solution step by adopting methanol and fixing the volume to prepare a series of reference substance solutions with different concentrations.
In a series of control solutions with different concentrations, the concentration ranges of liquiritin, hesperidin, honokiol and magnolol are the same as the step 2 in the example 1.
3. Measurement of
Respectively detecting the sample solution 3# and a series of reference substance solutions with different concentrations by Ultra Performance Liquid Chromatography (UPLC), comparing retention time for qualitative determination, and quantifying by external standard method to obtain the concentrations of liquiritin, hesperidin, honokiol and magnolol in the sample solution 3#. The specific quantitative procedure was the same as in step 3 of example 1.
Wherein, the high performance liquid chromatography comprises the following detection conditions:
the detector is a Diode Array Detector (DAD); the chromatographic column is a WatersAcquity UPLC BEH C18 chromatographic column (the column length is 100mm, the inner diameter is 2.1mm, and the particle size of the filler is 1.7 mu m); the detection wavelength is 230nm; the column temperature was 45 ℃; the flow rate is 0.3mL/min; the sample injection amount is 2 mu L; the mobile phase is acetonitrile-0.05% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.05% phosphoric acid water solution; the analysis time is 34min; gradient elution.
The procedure for gradient elution was the same as in step 3 of example 1.
Example 4
A sample of the Zhengqi tablet extract is taken, and the sample solution is prepared by adopting the step 1 in the example 1. Meanwhile, negative samples without the taste of the vital qi tablet extractum of liquorice, dried orange peel and magnolia officinalis are respectively taken, and a negative sample solution is prepared by adopting the step 1 in the example 1. In addition, a reference solution is prepared according to the step 2 in the embodiment 1, wherein the content range of the liquiritin in the reference solution is 8 mug/ml, the content range of the hesperidin is 80 mug/ml, the content range of the honokiol is 8 mug/ml, and the content range of the magnolol is 8 mug/ml.
The test solution, the negative test solution and the reference solution are respectively measured according to the step 3 in the embodiment 1, the retention time is compared for qualitative determination, and the specific test results are shown in figures 1, 2 and 3. As can be seen from FIGS. 1, 2 and 3, 4 chromatographic peaks of the negative sample did not interfere with the test sample, and the specificity of the method was good.
Example 5
The detection method of liquiritin, hesperidin, honokiol and magnolol in the Zhengqi tablet extract sample is verified by methodology, and the performance index results are as follows.
1. Linear relation
An appropriate amount of liquiritin, hesperidin, honokiol and magnolol as reference substances is precisely weighed, and methanol is added according to the step 2 in the embodiment 1 to prepare a series of reference substance solutions with different concentrations. According to the chromatographic conditions of the step 3 in the embodiment 1, precisely sucking 1 μ L of reference solution, injecting the reference solution into an ultra-high performance liquid chromatograph, drawing a standard curve by taking the concentration of the reference as a horizontal coordinate (x axis) and the peak area of each index component as a vertical coordinate (y axis), and calculating to obtain a standard regression equation, a correlation coefficient, a linear range, a quantitative limit and a detection limit of liquiritin, hesperidin, honokiol and magnolol, wherein the standard regression equation, the correlation coefficient, the linear range, the quantitative limit and the detection limit are more than or equal to 10 and the detection limit is more than or equal to 3, and specific results are shown in a table 2.
As can be seen from Table 2, the linear relationship of the 4 index components in the respective sample introduction mass concentration ranges is good, which shows that the method of the invention has wide linear range and high accuracy.
TABLE 2
2. Stability of
Taking a healthy qi tablet extract sample of batch number 210901, preparing a test sample solution according to the step 1 in the example 1, performing sample injection analysis at 0h, 2h, 4h, 6h, 8h, 12h and 24h respectively according to the chromatographic conditions of the step 3 in the example 1, and recording chromatographic peak area data of liquiritin, hesperidin, honokiol and magnolol. The results show that the chromatographic peak areas of liquiritin, hesperidin, honokiol and magnolol are less than 1.71 percent within 24h, and that the test solution has no influence on the results and good stability in 24h detection.
3. Precision degree
Taking any reference substance solution prepared in the step 2 in the example 1, respectively and continuously injecting samples for 6 times according to the chromatographic conditions of the step 3 in the example 1, and recording the peak area data of liquiritin, hesperidin, honokiol and magnolol. The result shows that the peak area RSD of continuous 6 times of sample injection of 4 components is less than 0.24 percent, which indicates that the precision of the instrument is good.
4. Repeatability
Taking a vital qi tablet extract sample of the batch number 210901, precisely weighing 6 parts, preparing 6 parts of test sample solution in parallel according to the step 1 in the example 1, measuring according to the chromatographic conditions of the step 3 in the example 1, recording peak area data of liquiritin, hesperidin, honokiol and magnolol, and calculating the content. The results show that the RSD content of 4 index components is less than 0.91 percent, and the method has good repeatability.
5. Recovery rate of added standard
Weighing 9 parts of a normal gas tablet extract sample (batch number 210901) with known concentration, precisely weighing, respectively adding any reference substance solution (respectively equivalent to 50%, 100% and 150% of the original mass fraction) prepared in step 2 in example 1 with low, medium and high mass concentrations, taking 3 parts of each mass concentration, preparing a test sample solution according to step 1 in example 1, respectively carrying out sample injection analysis according to the chromatographic conditions of step 3 in example 1, and calculating the sample injection recovery rate and RSD of each component according to the measured amount and the added amount, wherein the results are shown in Table 3. As can be seen from Table 3, the average recovery of the 4 components was 98.72-99.70%, and the RSD was 0.97-1.92%, indicating that the accuracy of the process was good.
Table 3 test results of recovery with addition of a label (n = 3)
Example 6
25 batches of vital qi tablet extract samples of different batches are taken, a test sample solution is prepared according to the step 1 in the example 1, sample injection analysis is respectively carried out according to the chromatographic conditions of the step 3 in the example 1, the contents of liquiritin, hesperidin, honokiol and magnolol are respectively calculated, and the result is shown in a table 4.
As can be seen from table 4, 14 batches of 2021-year healthy qi tablet extracts and 11 batches of 2022-year healthy qi tablet extracts are determined by the established healthy qi tablet extract content determination method, and the total is 25 batches, from the results, the two years of glycyrrhizin and hesperidin in the 4 components have no significant difference, but the content difference of honokiol and magnolol is large, which indicates that the influence of the magnolia officinalis medicinal material on the healthy qi tablet extracts is large, and the follow-up production needs to pay attention to the purchase of the magnolia officinalis medicinal material.
TABLE 4 results of content measurement of samples
Compared with the method in the year 2021, ## P<0.01， ### P<0.001
in conclusion, the method for measuring the contents of various components in the extract of the qi-strengthening tablet provided by the invention has the advantages of good linear relation, good repeatability, good precision, high accuracy and good stability, can be used for accumulating data of multiple batches of samples, establishes a reasonable content limit, and ensures stable production and clinical curative effect. Therefore, the invention effectively overcomes various defects in the prior art and has high industrial utilization value.
The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.
Claims (10)
1. A method for measuring the contents of various components in an Zhengqi tablet extract comprises the following steps: dissolving the extract of the Zhengqi tablet in a solvent, performing ultrasonic extraction and filtration, detecting the obtained test solution by using ultra-high performance liquid chromatography, and determining 4 index components in the test solution: content of liquiritin, hesperidin, honokiol, and magnolol.
2. The method for determining the contents of multiple components in Zhengqi tablet extract according to claim 1, wherein the solvent is methanol.
3. The method for determining the contents of a plurality of components in a vital qi tablet extract according to claim 1, wherein the ratio of the mass of the vital qi tablet extract to the volume of the solvent is 1.
4. The method for determining the contents of a plurality of components in a vital qi tablet extract according to claim 1, wherein the ultrasonic extraction time is 20-40 minutes; and/or the power of the ultrasonic extraction is 200-300W; and/or the frequency of the ultrasonic extraction is 30-50 kHz; and/or controlling the water temperature in the ultrasonic instrument to be less than or equal to 30 ℃ before the ultrasonic extraction, and controlling the water temperature in the ultrasonic instrument to be less than or equal to 45 ℃ after the ultrasonic extraction.
5. The method for determining the contents of various components in Zhengqi tablet extract according to claim 1, wherein the detection by using ultra-high performance liquid chromatography comprises the following steps:
1) Preparing a reference solution: adding solvent to control of liquiritin, hesperidin, honokiol and magnolol, dissolving, and fixing volume to obtain control solution;
2) Sample detection: respectively detecting the test solution and the reference solution in the step 1) by adopting an ultra-high performance liquid chromatography, comparing the retention time, performing qualitative determination, and determining the content of liquiritin, hesperidin, honokiol and magnolol in the test solution by adopting an external standard method.
6. The method for determining the contents of multiple components in vital qi tablet extract according to claim 5, wherein in step 1), the content range of liquiritin in the reference solution is 0.90-45.22 μ g/ml, the content range of hesperidin is 4.75-237.61 μ g/ml, the content range of honokiol is 0.99-49.60 μ g/ml, and the content range of magnolol is 0.91-45.40 μ g/ml; and/or the solvent is methanol.
7. The method for determining the contents of a plurality of components in a vital essence tablet extract according to claim 5, wherein in the step 2), the detection conditions of the ultra-high performance liquid chromatography are as follows: the detector is a diode array detector; the chromatographic column is a C18 chromatographic column; the detection wavelength is 220-230 nm; the mobile phase is acetonitrile-0.05-0.15% phosphoric acid water solution, wherein, the phase A is acetonitrile, and the phase B is 0.05-0.15% phosphoric acid water solution; the analysis time is 34min; gradient elution.
8. The method for determining the contents of a plurality of components in Zhengqi tablet extract according to claim 5, wherein in step 2), the detection conditions of the ultra-high performance liquid chromatography further comprise: the column temperature is 30-50 ℃; the flow rate is 0.1-0.3 mL/min; the sample amount is 0.5-5 mu L.
9. The method for determining the contents of multiple components in the vital qi tablet extract according to claim 7, wherein the specific procedure of gradient elution is as follows:
0-5min, phase A: the volume ratio of the phase B is 17:83-17:83;
5-7min, phase A: the volume ratio of the phase B is 17:83-20:80;
7-11min, phase A: the volume ratio of the phase B is 20:80-40:60, adding a solvent to the mixture;
11-12min, phase A: the volume ratio of the phase B is 40:60-55:45, a first step of;
12-22min, phase A: the volume ratio of the phase B is 55:45-58:42;
22-23min, phase A: the volume ratio of the phase B is 58:42-95:5;
23-28min, phase A: the volume ratio of the phase B is 95:5-95:5;
28-29min, phase A: the volume ratio of the phase B is 95:5-17:83;
29-34min, phase A: the volume ratio of the phase B is 17:83-17:83.
10. use of the method for determining the content of a plurality of components in a vital essence tablet extract according to any one of claims 1 to 9 in quality detection of the vital essence tablet extract.
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